Journal of General Microbiology (1991), 137, 1503-1509.
Printed in Great Britain
1503
Fatty acid composition of the estuarine Flexibacter sp. strain Inp: effect of
salinity, temperature and carbon source for growth
P.INTRIAGO*t and G . D. FLOODGATE
School of Ocean Sciences, Marine Science Laboratories, Menai Bridge, Gwynedd LL59 5E Y, UK
(Received 2 October 1990; revised 25 January 1991 ;accepted 6 March 1991)
The total fatty acid content of the estuarine Ffexibactersp. strain Inp and the relative proportions of its constituent
fatty acids were affected by growth temperature and salinity. Whilst both the proportion and concentration of the
polyunsaturates were markedly stimulated by increases in salinity, the total amount of fatty acid per mg cell
protein decreased. The highest concentration of fatty acid per mg cell protein did not coincide with the highest
percentage of polyunsaturated fatty acids, which occurred when the bacterium was grown on glucose. The presence
of an inverse relationship between C16:lw5 and C18:lw9 are regarded as evidence that two Merent pathways
exist for the biosynthesis of unsaturated fatty acids in FZexikter strain Inp.
Introduction
Changes in temperature, salt concentration and carbon
source in the medium are known to produce variations in
the membrane lipids of bacteria (Marr & Ingraham,
1962; Okuyama et al., 1977; Kawagushi & Seyama,
1984; Russell & Kogut, 1985; Kates, 1986a; Rose, 1989).
Variations in fatty acid composition due to changes in
temperature and salinity differ between Gram-negative
and Gram-positive bacteria. For example, lowering
'the growth temperature of the Gram-positive halotolerant Planococcus sp., results in increased amounts of
branched and monounsaturated fatty acids (MUFAs),
while the concentration of aC15 : O increases as the salt
content is raised (Miller, 1985, 1986). In Deleya halophila,
a moderately halophilic, Gram-negative bacterium,
there is a decrease in MUFAs with a concomitant
increase in cyclopropane fatty acids as the salt concentration in the medium is raised (Monteoliva-Sanchez et al.,
1988).
Reports on the fatty acid composition of members of
the genus Flexibacter vary. Fautz et al. (1979) and Poen et
al. (1984) reported branched as well as hydroxy fatty
acids to be the main components. Nichols et al. (1986),
working with several species, reported monounsaturated
and branched fatty acids as the most important groups,
t Present address: Aqualab, PO Box 5738, Guayaquil, Ecuador.
Abbreviations: FAME, fatty acid methyl ester; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; p.p.t., parts per
thousand (salinity).
0001-6543 O 1991 SGM
and Johns & Perry (1977) reported the presence of high
amounts of long chain polyunsaturated fatty acids
(PUFAs) in F. polymorphus.
PUFAs have been considered to be absent from
bacteria (Shaw, 1974). However, recent work on bacteria
from the marine environment has indicated that PUFAs
may be more widespread than thought hitherto. Delong
& Yayanos (1986) and Wirsen et al. (1987) examined a
number of deep-sea bacteria and found significant
amounts of PUFAs, as did Johns & Perry (1977) working
with the marine species F. polymorphus. More recently,
Yazawa et al. (1988) showed that 1.6% of isolates from
fish intestines produced C20 :5w3 ; in one strain, which
was similar to the genus Alteromonas, this compound
comprised 36% of the total fatty acids. The ecological
role played by PUFA-producing bacteria in the sea
remains obscure, but it is possible that they contribute to
the PUFA intake of larval forms including commercially
important crustacean species.
The bacterium Flexibacter sp. strain Inp, used in this
study, is widely distributed in the Ecuadorian shrimp
ponds from which it was isolated. These ponds are
subjected to wide variations in temperature and salinity.
Flexibacter strain Inp is orange pigmented and forms
filamentous threads with spreading growth on plates ;
gliding is not very active. It can utilize nitrate and
glutamate as nitrogen sources and peptone can be used as
both carbon and nitrogen source. It can degrade gelatin
and starch, but not agar or alginate. It is sensitive to
lauryl sulphate, does not survive in fresh water, but grows
on sea water media, tolerating up to 120 p.p.t. salinity. It
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1504
P . Intriago and G . D . Floodgate
is catalase- and oxidase-positive. Reichenbach (1989), in
the most-recent edition of Bergey's Manual of Systematic
Bacteriology, suggests that marine Flexibacter-like
strains should be considered as members of the genus
Microscilla.
Methods
Culture techniques. Cultures were maintained at 25 "C on ZoBell
2216-E solid medium (Oppenheimer & ZoBell, 1952). Before starting
each experiment a subculture, taken from a 1- or 2-weeks-old ZoBell
culture plate, was suspended in the medium to be tested; with the
exception of the carbon source, this medium consisted of 2 g
ammonium sulphate, 0.2 g dipotassium hydrogen phosphate, 0.5 g yeast
extract, 0.5 g Tris/HCl and 1 ml of trace metal solution (Reichenbach &
Dworkin, 1981) in 1 litre of sea water of salinity 30 p.p.t., adjusted to
pH 7-6. Cycloheximide (0.1 g 1-I) was used to inhibit growth of
eukaryotes. Since sea water contains a considerable number of
different ions, salinities are quoted in the oceanographic sense, as parts
per thousand (p.p.t.) (Riley & Chester, 1971). Salinities were measured
with an inductive salinometer.
Temperature experiments. The temperatures tested were 18, 24 and
30 "C. Experiments were done in 250 ml flasks with 50 ml of culture
medium and 5 ml of inoculum. Cultures were bubbled with 200: 10 ml
of an air/nitrogen mixture min-'. The flasks were immersed within a
water/bath controlled at the appropriate temperature.
Salinity relationships. Experiments were done using either starch or
ZoBell2216-E medium as carbon source, at 15,30,45 and 60 p.p.t., or
30, 45, 60 and 120 p.p.t., respectively. Cultures were bubbled with
100 :20 ml of an air/nitrogen mixture min-*, and incubated at 24 "C in
a water-bath. Salinity was decreased by mixing the sea-water medium
(salinity 30 p.p.t.) with distilled water, or increased by adding the
mixture of marine salts described in Reichenbach and Dworkin (1981).
Inclusion of betaine (10 mM) in the medium was necessary to obtain
growth of strain Inp at high salinities (45, 60 and 120 p.p.t.). Betaine
and related compounds are known to promote growth of bacteria in
media of high osmotic strength (Le Rudulier & Bouillard, 1983;
Imhoff & Rodriguez-Valera, 1984; Le Rudulier et al., 1984).
Carbonsources. A mixture of sterile air/nitrogen (100 :20) was used to
aerate the cultures. The salinity of the medium was 30 p.p.t. and the
temperature ws 24 "C. Except for the carbon source and the exclusion
of yeast extract, the medium used was as described above. The carbon
sources and their concentrations were as follows : starch, glucose,
lactose, gluconate and Casamino acids (5.0 g PI); glycerol (3.9 ml l-l);
Tween 80 (1 2.6ml l-I). Except for Casamino acids and Tween 80, these
values were chosen to normalize the carbon :nitrogen ratio of the
medium to 5 : 1. ZoBell 2216-E medium diluted to half-strength
(1/2-ZoBell) by the addition of sea water was also used.
Cultures were harvested during the late exponential phase, which
ranged from 25 to 50 h depending on temperature, salinity and the
carbon source used, by centrifugation at 10000g for 15 min at 10 "C;
the bacteria were then washed once in NaCl (0.5 M) and centrifuged
again. The bacterial pellet was resuspended in 10ml of potassium
phosphate buffer (50 mM, pH 7.5) (White et al., 1979) and stored at
- 20 "C.
Lipid extraction. Lipids were extracted by adding enough chloroform/methanol (1 :2, v/v) to a thawed cell pellet to obtain a single phase
chloroform/methanol/phosphate buffer mixture (1 :2 :0.8, by vol.).
This extract was left at 4 "C overnight, then water and chloroform were
added to form two separate phases (Bligh & Dyer, 1959; White et al.,
1979). The total cell fatty acids, present in the lower layer, were
extracted using a technique based on that of Moss et al. (1974) and
Larsson & Odham (1984). The lipid exract was dried under a stream of
nitrogen and then saponified with either 5% (w/v) NaOH or KOH in
50% (v/v) methanol at 100 "C for 45 min. The methanolysates were
cooled, and adjusted to pH 2 with HCl (6 M). Chloroform (1 ml) was
added and the mixture was shaken vigorously and allowed to separate
into two phases. The upper phase was discarded and the lower fattyacid-containing phase was dried under a stream of nitrogen. The fatty
acids were esterified with 2 ml 10% (v/v) BC13 in methanol at 80 "C for
10min. After cooling, the fatty acids methyl esters (FAMEs) were
extracted by adding 1 ml saturated NaCl and 2 ml hexane/chloroform
(4: 1, v/v). The extract was evaporated under a stream of nitrogen,
redissolved in chloroform and spotted on to a TLC plate (precoated
silica gel 60, Merck) to separate FAMEs from other reaction products.
The plates were developed for 90 min in a continuous elution tank using
hexane/diethyl ether (90 : 10, v/v) as the solvent system. FAMEs were
located using iodine vapour and areas of silica gel containing FAMEs
were scraped from the plates and the FAMEs dissolved in chloroform/methanol (2 : 1, v/v). The solvent was evaporated to dryness and
the FAMEs were redissolved in 2 0 0 ~ 1hexane.
FAMEs were analysed using a Carlo Erba gas chromatograph (model
Vega 6180) with on-column injection into a 30 m x 0.25 mm i.d. DB225 fused silica column (J & W Scientific) or an Econo-cap, Carbowax
30 m x 0.25 mm i.d. capillary column (Alltech). Hydrogen was the
carrier gas (2.0 ml min-I). The oven temperature was programmed to
rise from 50 to 150 "C at a ramp rate of 49-9 centrigrade degrees min-'
and then 3 centigrade degrees min-' to 230 "C, and held for 10 min.
The temperature was 250 "C. Uniform response was assumed for all
components. Quantification was based on comparison of peaks areas
with an internal standard (C23:O). FAMEs were identified by
comparison of their retention times with known standards. The
identity of PUFAs was confirmed by GC/MS using a Finnigan gas
chromatograph/mass spectrometer fitted with a 30 m x 0.25 mm i.d.
OV1 capillary column, and with helium as the carrier gas. Samples
were injected in splitless mode at 180 "C. The oven temperature was
programmed to rise from 60 to 280 "C at a ramp rate of 6 centrigrade
degrees min-'. The electron impact ionization voltage was 70 eV.
Hydrogenation was also used to confirm the presence of double bonds
(Kates, 19863), whose position was determined by MS analysis of the
Diels-Alder adducts, formed by reacting FAMEs with 5,Sdimethoxy1,2,3,4-tetrachlorocyclopentadiene (Aldrich) (Kidwell & Bieman,
1982; Nichols et al., 1985).
Protein. Protein was determined by a modification of the method
of Lowry, after extracting the cells in NaOH (1 M)(Hanson & Phillips,
1981). Bovine serum albumin was the standard used.
Chemicals. All reagents used were analytical grade (Sigma and
BDH). Solvents were from Rathburn.
Statistics. Analyses were done using the Minitab statistical package
on a Vax mainframe computer.
Gas chromatographylmass spectrometry
The mass spectra of commercial standards of linoleic and
linolenic acid were found to be identical to that of the
compounds found in Flexibacter strain Inp, and identified as linoleic and linolenic acids from their retention
times. The ion fragments of both the C16:lw5 and
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Fatty acids in Flexibacter ‘strain Inp
1505
100-
100 -
409
n
5x
.I
C
5!z
v)
.I
h
E
-
x
-
v)
E
2-50-
50-
I ,i’ t
0
.I
c1
2
&
-
C
.I
-
100
150 200
250
300
350 400
.d
0)
.d
c,
d
c2
-
75
I
28 I
100
450 500 550
200
300
400
500
600
mlz
mlz
Fig. 1
Fig. 1. Mass spectrum of the Diels-Alder adducts of C16:lw5.
Fig. 2. Mass spectrum of the Diels-Alder adducts of C18:lw9.
Fig. 2
Table 1. Fatty acid composition of strain Inp afer growth at diflerent temperatures
The composition is expressed as a weight percentage of the total fatty acids. The results
presented are means of two experiments at each temperature; values in parentheses represent
+1 SD.
~
~~
Fatty acid composition (%)
Growth temp. (“C) . . .18
Fatty acid
14:O
i15:O
a15:O
15:O
16:O
16: lw9
16: lw7
16: lw5
Unknown
Unknown
17 : O
18 : O
18:lw9
18 : lw7
18:2w6
18:3w3
Total fatty acid content
[pg (mg protein)-’]. . .
Percentage unsaturation . . .
Unsaturation index*. . .
* Unsaturation
index =
A‘[ monounsaturates)
C18 :lw9 Diels-Alder adducts confirmed unequivocally
the identificationof the two main monounsaturated fatty
acids in strain Inp (Figs. 1 and 2).
Eflect of temperature
As shown in Table 1, C16 : lw5 was the major fatty acid
found under all conditions, ranging from 48.1 to 67.7% of
24
30
1-15(0.88)
0.53 (0.55)
5-00 (1.48)
3.09 (2-02)
0.16 (0.21)
0.29 (0-07)
0-26(0.36)
0.49 (0.08)
11.96 (2.97) 14.92 (4.70)
3-03(0.70) 1.58 (2.23)
0.41 (0.57)
53.80 (2.70) 48.10 (0.33)
0.39 (0.55)
1-00(1.42)
0.06 (0.08)
0.49 (0.21)
0.06 (0.07)
0.40(0.09)
2-50 (0.96)
2-74(1.00)
9.40 (1.54) 10.40 (0.39)
0.98 (1.40)
3.26 (3.90)
8.72 (2.51) 11.74 (1.10)
1.07 (0.30)
0.45 (0.35)
1.69 (0.05)
8.68 (2.98)
0.93 (1.30)
0.25 (0.35)
18.40 (1.85)
0.03 (0.04)
0.70 (0.98)
49.01 (0.00)
1.26 (1.74)
0.10 (0.15)
1.06 (0.98)
4.50 (2.18)
5-82 (1.74)
1-15(1-62)
4.69 (1.96)
0.14 (0.20)
70.8 (5.4)
56.6 (4.6)
74.3 (5.5)
78.2 (1.8)
0.88 (0.04) 0.88 (0.07)
158 (22)
61.5 (3.7)
0.66 (0.03)
+ 2(% diunsaturates) + 3(% triunsaturates)]/ 100.
the total fatty acid content. Palmitic acid (C16 :0) also
formed a significant fraction of the total. Oleic acid
(C18 : lw9), and linoleic acid (C18 :2w6) were next in
order of abundance. The PUFAs linoleic (C18 :2w6) and
linolenic acid (C18 : 3w3) ranged from 4.7 to 1 1.7% and
0.1 to 1.1% respectively. The degree of unsaturation,
measured as percentage unsaturation and unsaturation
index, tended to decrease with increasing temperature
(Table 1).
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1506
P. lntriago and G . D. Floodgate
Table 2. Fatty acid composition of strain Inp afier growth at diflerent salinities on
starch as carbon source
The composition is expressed as a weight percentage of the total fatty acids. The results
presented are means of three experiments at each salinity; values in parentheses represent
1 SD.
Fatty acid composition <%)
Fatty acid
Salinity (p.p.t.) . . .15
30
45
60
0-60 (0.20)
1.27 (0.41)
0.33 (0.36)
0.22 (0.09)
10.40 (3-63)
2.60 (2.27)
5.12 (3.70)
19.01 (2.88)
0.23 (0.39)
3.52 (1.74)
26.70 (3-06)
6.30 (1.71)
20.45 (3.90)
1 a90 (0.70)
3 1 *6(3.08)
~~
14:O
i15:O
a15:O
15:O
16:O
16: lw9
16: lw7
16: lw5
Unknown
Unknown
17:O
18 :O
18:lw9
18:lw7
18:2w6
18:3w3
Total fatty acid content
[pg (mg protein)-'] . . .
1.00 (0-26)
0.69 (0.16)
9.31 (1-70)
3.72 (0.50)
2-14 (1.50)
2.76 (0.50)
0.39 (0-12)
0.19 (0.01)
8.57 (2.15)
7-22 (2-01)
1.36 (0.40)
2.26 (1.79)
2.56 (2.23)
5.22 (2.96)
65.30 (2.62) 69.00 ( 1.10)
0-32 (0.55)
0.20 (0.34)
0.23 (0.28)
0.39 (0.14)
2.68 (0.29)
0.55 (0.05)
2.23 (0.58)
0.31 (0.09)
0.26 (0.08)
2.09 (1-07)
0.73 (0.24)
2.30 (0.93)
0.20 (0.08)
0.67 (0.19)
3.26 (0.57)
1 *32(0.53)
0.29 (0.30)
9.99 (2.23)
2.14 (2.33)
1.50 (1.01)
44-07 (5.55)
0.21 (0.38)
0.05 (0.09)
0-08 (0-14)
1.84 (0.88)
13.88 (4.1 3)
6-38 (2.14)
1 1.44 (0.88)
0.57 (0.41)
119.6 (10.0)
81-28 (23-0)
38.5 (10.3)
-
-
-
0.44 (0-38)
Table 3. Fatty acid composition of strain Inp afier growth at diferent salinities on
112-ZoBell medium
The composition is expressed as a weight percentage of the total fatty acids. The results presented are
means of three experiments at each salinity; values in parentheses represent k 1 SD.
Fatty acid composition (%)
Fatty acid
14:O
i15:O
a15:O
15:O
15:1*
16:O
16: lw9
16: lw7
16: lw5
Unknown
Unknown
Unknown
17:O
18 :O
18:lw9c
18 : lw9t
18:lw7
18:2w6
18:3w3
Total fatty acid content
[pg (mg protein)-'] . . .
Salinity (p.p.t.). . .30
45
60
120
1-18(0.06)
17.67 (0.96)
0.29 (0.07)
0.67 (0.00)
1.93 (1.72)
4.18 (0.40)
0.87 (0-37)
0.37 (0.21)
55.49 (2.50)
0.44 (0.03)
0.81 (0.02)
0.94 (0.04)
0.09 (0.09)
0.78 (0.12)
5.88 (1.10)
0.81 (0.27)
0.66 (0.33)
5.06 (1 -09)
0.47 (0.09)
1a25 (0.04)
20.95 (1.58)
0.33 (0.10)
0.58 (0.04)
1.38 (0.12)
4.11 (1.58)
1.31 (0.68)
0.1 5 (0.25)
49.90 (2-92)
0-24 (0.09)
1.57 (0.10)
0.86 (0.18)
0.15 (0.08)
1.08 (0.58)
6.04 (0.92)
0.95 (0.22)
0.80 (0.1 1)
4.91 (0.53)
0.47 (0.10)
1.54 (0.22)
14.20 (1.40)
0-20 (0.1 1)
0.55 (0.03)
0.72 (0.08)
11.80 (0.61)
1.58 (1.61)
0.22 (0.22)
53.79 (2.64)
0.14 (0.05)
1-56(0.1 1)
0.27 (0.03)
0.20 (0.02)
1.50 (0.28)
4.57 (0.42)
0.70 (0.1 1)
0.47 (0.05)
3.63 (0.10)
0.70 (0.1 1)
0.79 (0.08)
0.98 (0.34)
0.10 (0.10)
0.11 (0.10)
0.14 (0.13)
22.99 (1-54)
0.19 (0.16)
0.35 (0.31)
4-75 (2.69)
56.7 (17.9)
34.7 (2.50)
30.9 (9.66)
7-7 (0.02)
* Double bond
position not determined.
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-
-
0.08 (0.14)
0.17 (0.15)
5.68 (0.41)
27-14 (4.17)
4.16 (0.30)
3.53 (0.64)
22-35 (3.52)
2.01 (0.50)
Fatty acids in Flexibacter strain h p
1507
Table 4. Fatty acid composition of strain Inp after growth on diferent carbon sources
The composition is expressed as a weight percentage of the total fatty acids. The results presented are means of three experiments (for Casamino acids,
Tween 80, glycerol, glucose and starch) or of two experiments (for lactose and gluconate); values in parentheses represent k 1 sD.
Fatty acid composition (%)
Fatty acid
Carbon source. . . Casamino acids
14:O
i15:O
a15 : O
15 : O
15:1*
16:O
16: lw9
16: lw7
16:lw5
Unknown
Unknown
Unknown
i17:O
a17:O
17:O
18:O
18:lw9c
18:lw9t
18 : lw7
18:2w6
18:3w3
Total fatty acid content
[pg (mg protein)-']. . .
1.09 (0.04)
12.47 (1 ~24)
0.52 (0.16)
0.66 (0.03)
0-68 (0.01)
6.43 (0-10)
2.86 (1*49)
0.50 (0.20)
35.18 (1.80)
1.79 (0.54)
0.99 (0.18)
0.74 (0.07)
Tween 80
Glycerol
Glucose
Starch
Lactose
Gluconate
3.30 (0.49)
3.1 7 (0.45)
0.29 (0.01)
0.78 (0.08)
0.20 (0.20)
19-17(2.70)
1.44 (0.18)
4.60 (0.37)
17.90 (3.94)
1*02(0.22)
0.08 (0.13)
0.50 (0.07)
1.27 (0.09)
10.81 (1.42)
0.65 (0.15)
1.00 (0.26)
0.75 (0.32)
7.69 (0.78)
1.55 (0.60)
0.48 (0.10)
34.61 (0.72)
0.65 (0.27)
1.00 (0.71)
0.80 (0.41)
0.27 (0.00)
0.55 (0.02)
3.34 (0.37)
0.11 (0.1)
tr
0.11 (0.19)
18.02 (2.26)
0.65 (0.27)
0.11 (0.19)
30.11 (2.5)
0.51 (0.07)
1.76 (0.23)
8.5 (0.92)
0.78 (0-33)
0.60 (0.05)
0.30 (0.04)
13.77 (2-47)
0.54 (0.00)
3.34 (0.68)
0.16 (0.05)
0.19 (0.05)
1.18 (0-06)
8.24 (0.36)
1.37 (0.60)
2.40 (0.55)
1.05 (0.10)
12.42 (1-07)
3.10 (2.25)
-
-
0.14 (0.12)
0.17 (0.16)
1.82 (0.09)
12.13 (1.06)
1.93 (0.07)
1-52(0.17)
10.53 (0.17)
0.97 (0.08)
-
40.0 (6.24)
-
-
-
3.48 (1.92)
60.15 (2-85)
0.53 (0.47)
0.51 (0.1 1)
0.30 (0.07)
-
0.08 (0.13)
0.78 (0.1 7)
1.72 (1.18)
-
43.6 (15.2)
64.8 (10.1)
43.9 (0.50)
-
~~~
* Double bond position
Efect of salinity
Increase in salinity was associated with a marked
decrease in the total fatty acid content per mg cell protein
(Tables 2 and 3). Both iC15 :O and C16 : lw5 decreased
markedly as salinity increased, while C18 : lw9 and
PUFAs increased. Linoleic and linolenic acid ranged
from 2.2 to 22.4%and 0.2 to 2.0% respectively. Analysis
of variance showed that both the proportions and the
concentrations of the major fatty acids were significantly
different over the range of salinities tested (P>O-l).
Increasing the salinity from 30 to 120 p.p.t. in 1/2-ZoBell
medium resulted in a marked decrease in iC15 :O with a
concomitant increase in C16 :O. The percentage of
C 16 : 1w5, the major fatty acid in strain Inp, was constant
at salinities from 30 to 60 p.p.t., but at 120 p.p.t. it
decreased from about 60% to 4%, whereas both oleic
acid and PUFAs increased markedly.
Eflect of carbon source
The highest concentration of fatty acid per mg cell
protein was found in cells grown on starch (Table 4)
followed by cells grown on gluconate or glycerol.
-
-
0.07 (0.1 1)
3.23 (1.1 1)
16.08 (1.16)
2-25 (0.89)
2.60 (0.40)
17.77 (1*26)
2.25 (0.89)
4.02 (0.86)
1*42(0.25)
-
-
0.03 (0.06)
1.88 (0.40)
14.92 (2.80)
1.98 (0.34)
1-97(0-29)
12.07 (1.47)
1-14(0.15)
0-44(0.10)
4.65 (1*97)
29.03 (3.96)
-
20.40 (0.23)
0.13 (0.18)
0.23 (0.18)
59.94 (2.56)
-
-
57.62 (2.32)
0.35 (0.02)
1.51 (0.12)
0.47 (0.08)
0.44 (0.10)
-
0.1 7 (0.07)
1-14(0.70)
0.22 (0.03)
1a08 (0.25)
4.40 (1-77)
0.72 (0.27)
0.61 (0.20)
2.77 (0.10)
0.22 (0.05)
0.83 (0.08)
3.31 (0.12)
0.54 (0.02)
0.51 (0.00)
3-11 (0.25)
0.24 (0.07)
78.2 (3-50)
64.5 (1.36)
52-8 (13.1)
-
~
not determined.
Both the highest concentration per mg cell protein and
the highest percentage of PUFAs were found in cells
grown on glucose, glycerol or Casamino acids (Table 4).
iC15 :0, the main branched fatty acid present in strain
Inp, was found in especially high concentrations in cells
grown on Casamino acids. The concentration and
proportion of C 16 :1w5 were inversely proportional to
those of PUFAs. The fatty acid composition of strain Inp
grown on Tween 80 was influenced by the presence of the
dominant fatty acid in the growth medium.
Discussion
In general, the fatty acid composition of Flexibacter
strain Inp resembles that found by Nichols et al. (1986)
for several Flexibacter species. These authors found that
iC15:0, C16:O and MUFAs with carbon chain lengths
of either 16 or 18 comprised the bulk of the fatty acids.
The position of the double bond in the monounsaturates
and the presence and relative importance of branched
fatty acids varied between species. Amongst the species
examined by these authors F . Jexilis, which has
C16: lw5 as its major fatty acid, is perhaps the most
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1508
P . Intriago and G . D . Floodgate
closely related to strain Inp. Reports of C16:lw5 in
micro-organisms are uncommon, although it has been
found in Cytophaga hutchinsonii (Walker, 1969) and in F.
pofymorphus (Johns & Perry, 1977). The differences in
the proportions of the major fatty acids between F.
jlexifis and strain Inp could be due to differences in the
composition of the growth media used. In their experiments, Nichols et a f . (1986) grew F. Jexilis in a proteinrich medium. It is known that branched fatty acids can
be synthesized from branched amino acid precursors
(Boulton & Ratledge, 1985). In addition, F.jlexilis is a
fresh-water species. It is not uncommon to find differences in fatty acid composition between fresh-water and
marine species of the same genus (Wood, 1974).
There is an apparent discrepancy in the fatty acid
compositionsof strain Inp as determined in experiments
in which temperature or salinity were varied. This
difference could be due to the different air/nitrogen
bubbling rates used in the two types of experiment.
The drop in fatty acid yield per unit protein as salinity
increased is similar to that found by Pugh et a f . (1971),
who suggested that a high salt concentration could
inhibit fatty acid synthetase. it is interesting to note that
whereas cells of strain Inp grown on 1/2-ZoBell or on
starch medium contained similar concentrations of fatty
acid per mg cell protein at 60 p.p.t. salinity, the
proportion of PUFAs in cells grown on starch medium at
60 p.p.t. salinity was similar to that in cells grown on 1/2ZoBell at 120 p.p.t. This suggests that the possible
inhibition of fatty acid synthesis by increasing salt
concentration in strain Inp was not directly coupled to
the stimulation of PUFAs by increasing salinity.
The fatty acid composition of strain Inp when fatty
acids present in Tween 80 were used as the carbon source
reflected the major fatty acid present in the medium.
Analysis of Tween 80 showed that 80% of its total
fatty acid was C18 : lw9. The absence of PUFAs from
strain Inp when using Tween 80 as carbon source could
be due to repression of the fatty acid synthesis enzymes
by the free fatty acids present in the culture medium.
This occurs in many species of bacteria (Weeks & Wakil,
1970; Nunn, 1986; Schweizer, 1989).
Of the major fatty acids found in strain Inp, C18 : 1w9,
C18 :2w6 and C18 :3w3 are likely to be related by a
common biosynthetic mechanism as is found in green
algae and higher plants, namely the aerobic pathway for
synthesisof unsaturated fatty acids (Harwood & Russell,
1984). If strain Inp has only the aerobic pathway, then
two desaturases would be required, one specific for the
A9 carbon and another for the A1 1 carbon. The presence
of two desaturases has been reported in some bacteria
(Fulco, 1970, 1983). If two such enzymes are present in
strain Inp, the All and A9 desaturases are almost
exclusively specific for fatty acids with carbon chain
lengths of 16 and 18, respectively. Additionally, the
product of elongation of C16: lw5 would be either
C18 :lw5 or C18 : lw7, depending at which end the two
carbon atoms are inserted, but it is mostly likely to be
C18 : lw5. Hence the oleic acid could not originate from
C 16 : 1w5, Therefore, it is reasonable to hypothesize that
strain Inp possesses both the anaerobic and aerobic
pathways for synthesis of unsaturated fatty acids.
Although Scheuerbrand & Bloch (1962) suggested that
both pathways were mutually exclusive, more recently
Wada et al. (1989) have demonstrated the presence of
both pathways in a psychrotropic Pseudomonas.
The inverse relationship between the amounts in
strain Inp of the two major fatty acids, C16: lw5 and
C18 : lw9, both of which were identified unequivocally
by GC and GC/MS, can be explained in terms of a linked
flux mechanism so that when one pathway is dominant
the other is recessive, or vice versa.
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